Cytokine Regulation of Bone Cell Differentiation

VITAMINS AND HORMONES, VOL. 52

Cytokine Regulation of Bone Cell Differentiation MELISSA ALSINA," THERESA A. GUISE,? AND G. DAVID ROODMAN*,$ Department of Medicine, Divisions of *Hematology and +Endocrinology, University of Texas Health Science Center at Sun Antonio, and rAudie Murphy Veterans Administration Hospital, Sun Antonio, Texas 78284 I. Introduction 11. Osteoblasts A. General Characteristics of the Osteoblast B. In Vitro Systems to Study the Osteoblast C. Factors Involved in Proliferation and Differentiation 111. Osteoclasts A. General Characteristics of the Osteoclast B. Systemic Hormones That Affect Osteoclast Function and Formation C. Autocrine-Paracrine Factors with Osteoclast-Stimulatory Activity D. Local Inhibitory Factors IV. Summary References

I. INTRODUCTION The activity of bone cells-osteoblasts and osteoclasts-is under control of both systemic hormones and cytokines generated in the bone microenvironment. These factors may act directly on osteoblasts, osteoclasts, or their precursors, or may act indirectly through an intermediary cell to control the formation, differentiation, and function of bone cells. In this article, we review the effects of these factors on bone cell differentiation and function. 11. OSTEOBLASTS Bone formation is the result of a series of events that involve proliferation of early mesenchymal cells, differentiation into osteoblast precursor cells, maturation of osteoblasts, formation of matrix, and, finally, mineralization. The major processes leading to bone formation include recruitment and replication of mesenchymal precursors of osteoblasts that are found in the periosteum and in the bone marrow adjacent to endosteal surfaces, their differentiation from preosteoblast to osteoblast, and regulation of their activity. 63

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A. GENERAL CHARACTERISTICS OF THE OSTEOBLAST The osteoblast produces a wide variety of proteins that compromise the bone matrix, including type I collagen, osteocalcin, matrix Gla protein, osteonectin, alkaline phosphatase, fibronectin, thrombospondin, proteoglycans, osteopontin, and bone sialoproteins. The osteoblast also secretes growth factors such as transforming growth factor-p (TGF-P),bone morphogenetic proteins (BMPs),insulin-like growth factor-I and -11 (IGF-I, -II), platelet-derived growth factor (PDGF), and heparin-binding fibroblast growth factor (FGF), which are stored in bone matrix (Robey, 1989). These growth factors stimulate proliferation and differentiation of bone cells. Osteoblasts mineralize newly formed bone matrix, their presence appears to be required for osteoclastic bone resorption in rodents, and they enhance the bone-resorbing capacity of human osteoclasts. The osteoblast family also includes osteocytes and bone-lining cells. The function of these cells is not well understood, but they may play an intricate role in bone remodeling. TO STUDY THE OSTEOBLAST B. In Vitro SYSTEMS

The development of experimental models used to study the osteoblast phenotype and bone formation has led to a better understanding of the function of cells of the osteoblast lineage. In uitro culture systems utilizing freshly dispersed bone cells, osteosarcoma cell lines, and nontransformed osteoblast-like cell lines derived from normal calvarial cells have been used to study differentiation of these cells in response to different osteotropic hormones. Unfortunately, these cell lines are not useful for determining lineage, and the variability in hormone responsiveness among cell lines makes it difficult t o extrapolate such results to the in uiuo situation (Mundy, 1995a,b). Recently, an in uitro system of prolonged primary cultures of isolated fetal rat calvarial osteoblasts with P-glycerophosphate and ascorbic acid has been developed in which bone cells form clusters over several weeks and eventually become surrounded by woven bone (Bellowset al., 1986).There is an initial stage of bone cell proliferation and extracellular matrix (ECM) biosynthesis followed by bone ECM development, maturation, and organization. The last phase is ECM mineralization. A developmental cascade of gene expression can be demonstrated in such cultures of fetal rat calvarial cells grown over several weeks. Specifically, during the proliferative phase, histone (reflecting DNA synthesis), c-fos, c-jun, and type I collagen gene expression are maxi-

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mal. Histone, c-fos, and c-jun genes encode proteins that support proliferation of osteoblast precursors. Additionally, several genes associated with formation of the ECM, such as type I collagen, fibronectin, and TGF-P, are actively expressed during this proliferative phase of osteoblast development and then gradually down regulated during the subsequent stages of osteoblast differentiation. During the matrix maturation phase, when osteoblasts are postmitotic, alkaline phosphatase is expressed. The mineralization phase is characterized by maximal expression of the osteopontin, osteocalcin, and bone sialoprotein genes along with the accumulation of calcium (Stein and Lian, 1993). Thus, phenotypic markers of the differentiated osteoblast include alkaline phosphatase, osteopontin, and osteocalcin.

C. FACTORS INVOLVED IN PROLIFERATION AND DIFFERENTIATION 1. Systemic Factors a. Parathyroid Hormone. Parathyroid hormone (PTH) is a peptide produced by the parathyroid glands that is important in the maintenance of normal calcium homeostasis due to its stimulatory effects on osteoclastic bone resorption, renal tubular reabsorption of calcium, and production of la-hydroxylase activity in the kidney. When administered as a pharmacological agent, PTH has complex and different effects on bone depending on whether it is administered intermittently or continuously. Continuous infusion of PTH results in increased bone resorption. In contrast, administration of low doses of PTH in an intermittent fashion stimulates bone formation (Dempster et al., 1993). The primary target cell for PTH is the osteoblast (Rodan and Martin, 1981). Isolated osteoclasts do not resorb bone in response to PTH and only do so when primary cultures of osteoblasts or osteoblast-like cell lines are added (Chambers et al., 1985; McSheehy and Chambers, 1986). Recent studies have described a distinct PTH target cell that is located among clusters of differentiated osteoblasts that exhibited structural features of a large soma and long cytoplasmic processes. Since these cells stained positively for alkaline phosphatase and displayed ultrastructural features of the differentiated osteoblast, they appear to be in the osteoblast lineage (Rouleau et al., 1988,1990).More recently, PTH receptors have been demonstrated in osteoclasts and osteoclast precursors (Teti et al., 1991; Hakeda et al., 1989) and, in fact, PTH has been shown to inhibit bone resorption in isolated osteoclasts

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(Teti et al., 1991).Thus it appears that both osteoblasts and osteoclasts express functional PTH receptors and that PTH may have dual actions on bone: an indirect stimulatory effect mediated by osteoblasts and a direct inhibitory effect on the osteoclast (Dempster et al., 1993). In uitro, PTH inhibits collagen synthesis, alkaline phosphatase activity, and osteocalcin synthesis in cultured osteoblastic cells (Dempster et al., 1993).However, PTH stimulates replication of osteoblasts at low doses in cultures of clonal osteosarcoma cells (Martin et al., 1989) but inhibits osteoblast proliferation at high concentrations. In osteoblast-rich cultures, PTH promotes proliferation (Van der Plas et al., 1985). These observations suggest that the anabolic effect of PTH is mediated by an increase in proliferation of osteoblast precursors. In most systems, the mitogenic effect of PTH is associated with a reduction in the expression of the osteoblast phenotype. It should be remembered, however, that the response of different osteoblast cultures to PTH is variable and depends on whether they are primary cultures or a cell line, on the species and age of the source of the osteoblastic cells, and on the initial degree of differentiation in uitro. There is convincing evidence that PTH-stimulated bone formation is mediated by local growth factors such as IGF-I and -11and TGF-P. PTH stimulates production of IGF-I in isolated bone cells and in rodent calvarial organ cultures (Canalis et al., 1993). While PTH does not appear to regulate osteoblastic synthesis of TGF-P (McCarthy et al., 19891, its action to stimulate osteoclastic bone resorption results in liberation of TGF-P from bone matrix. The acidic microenvironment under the osteoclast may activate latent TGF-P, which would stimulate osteoblast chemotaxis, replication, and differentiation of osteoblasts. PTH also increases production of interleukin (1L)-6in bone cells (Feyen et al., 1989), and plasma IL-6 concentrations are increased in patients with primary and secondary hyperparathyroidism (Rusinko et al., 1995). b. Calcitriol. Calcitriol, or 1,25-&hydroxyvitamin D, (1,25-(OH),D3), is produced in the proximal tubules of the kidney by the la-hydroxylation of 25-hydroxyvitamin D, and is a potent bone-resorbing factor. 1,25-(OH),D3 increases intestinal absorption of calcium and phosphate, and its presence in uiuo is necessary for normal bone mineralization. The production and effects of calcitriol are intimately related to those of PTH as PTH stimulates production in the kidney of la-hydroxylase, the rate-limiting enzyme needed for the conversion of 25-hydroxyvitamin D3 to 1,25-(OH)2D3.Calcitriol receptors are located on osteoblasts and their precursors (Chen et al., 1983; Narbaitz et al., 1983). The effects of calcitriol on osteoblast-like cells in uitro are disparate and

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often do not correlate with what is known about its function in uiuo. In short-term bone organ cultures, calcitriol inhibits bone collagen synthesis (Raisz et al., 19801, and when incubated with cultured bone cells with the osteoblast phenotype, it increases expression of osteocalcin and alkaline phosphatase (Price and Baukol, 1980; Manolagas et al., 19811. In prolonged primary cultures of fetal rat calvarial cells, 1,25(OH),D, inhibited the formation of mineralized bone nodules, while in rodent osteoblasts and osteosarcoma lines, it inhibited osteoblast proliferation in a dose-dependent manner (Chen et al., 1983). In contrast, calcitriol stimulates DNA synthesis in periosteal cells of calvaria (Canalis and Lian, 1985). Thus, PTH and calcitriol can influence proliferation of osteoblasts or their precursors, and the effects may depend on the stage of differentiation of the target cell population tested, as well as the cell system used. c. Estrogen. Sex steroids play a n important role in bone physiology through the maintenance of mineral homeostasis and bone balance. Sex steroid deficiency predisposes to bone loss and fracture. Not only is estrogen the most important sex steroid in preventing osteoporosis in women, but the finding of functional estrogen receptor deficiency in a young male patient with osteoporosis and failure to fuse epiphyseal growth plates suggests that estrogen is important in maintaining bone mass in men as well (Smith et al., 1994). Estrogen receptors have been demonstrated in cultured bone cells of the osteoblast lineage (Komm et al., 1988; Eriksen et al., 1988) as well as on osteoclasts (Oursler et al., 1991b). The effects of estrogen on osteoblast-like cells, using a variety of in uitro models, have been conflicting (Turner et al., 1994). Estrogen has been reported to increase (Ernst et al., 19891, decrease (Gray et al., 19871, or have no effect on (Keeting et al., 1991) proliferation of cells of the osteoblast lineage from a variety of sources. Likewise, there are similar reports that estrogen increased (Gray et al., 1987), decreased (Watts et al., 19891, or had no effect on production of bone matrix proteins. It is likely that the differences in the model systems used to study the effects of estrogen on osteoblasts, and the fact that the cell culture systems do not mimic the environment in uiuo, may explain some of these conflicting results. This is in contrast to results in uiuo, when estrogen enhances bone formation. Estrogens stimulate production of TGF-P in human osteoblast-like cells (Oursler et al., 1991a). These effects may be mediated through the nuclear protooncogenes c-fos and c-jun (Turner et al., 1994; Weisz and %sales, 1990). There is sufficient evidence to suggest that estrogen has important

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effects on bone by modulating the production of locally active growth factors and cytokines in bone. Estrogen may decrease bone resorption by mediating release of cytokines and soluble growth factors by osteoblasts. This is likely a complex process and may involve multiple cytokines and growth factors. In osteoblast-like cells, estrogen has been shown to increase TGF-p production; have no effect on IL-lp, IL-8, or granulocyte-macrophage colony-stimulating factor (GM-CSF) production; and have variable effects on production of IL-6, IGF-I, IGF-11, and tumor necrosis factor (TNFI-(Y(Turner et al., 1994). d. Insulin. Insulin stimulates bone matrix synthesis and has a stimulatory effect on the differentiated function of the osteoblast. However, it does not increase the number of collagen-producing cells (Canalis, 1993).I n uiuo, insulin is necessary for the normal mineralization of bone. Patients with insulin deficiency and diabetes mellitus have a higher incidence of osteopenia, although the mechanisms responsible for this observation are not well understood. e. Growth Hormone. Growth hormone (GH) is an important stimulator of linear growth and also regulates bone formation after the growing phase of the skeleton is complete. Patients with GH deficiency have low bone mass, while patients with acromegaly often have increased bone mass. The mechanisms by which GH exerts its effects on bone are unclear. It stimulates production of IGF-I by skeletal cells, but it likely has other effects independent of IGF-I, as the anabolic effects of PTH on bone are not observed in hypophysectomized rats until GH is administered (Hock and Fonseca, 1990). f. Glucocorticoids. Glucocorticoids in excess profoundly decrease bone mass. Glucocorticoids inhibit gonadotropin and sex steroid production, and the resulting hypogonadism can result in osteopenia (Lukert and Raisz, 1990). In addition to these indirect effects, glucocorticoids inhibit bone formation and stimulate bone resorption in uiuo. I n uitro, glucocorticoids have complex effects on gene expression in osteoblasts. These actions are dependent on the stage of osteoblast growth and differentiation and on the cell model and culture conditions used. Glucocorticoids have been shown to induce cells of the osteoblast lineage to differentiate into mature cells expressing the osteoblast phenotype (Delany et al., 1994a). Other effects of glucocorticoid on bone include enhanced PTH receptor expression, decreased replication of preosteoblasts, inhibition of type I collagen and osteocalcin expression, increased interstitial collagenase expression, and decreased tissue inhibitor of metalloproteinases expression. Although glucocorticoids have no effect on TGF-P synthesis, they can shift the binding of TGF-Pl from signal-transducing receptors to

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non-signal-transducing receptors and decrease TGF-P mitogenic properties in bone cells (Centrella et al., 1992). Glucocorticoids also inhibit expression of IGF-1 but do not modify IGF-I1 synthesis by osteoblasts. These steroid hormones also modify IGF-binding protein (IGFBP) expression, another mechanism by which they can regulate IGF actions in bone. Six IGFBPs have been identified, and binding of these proteins to IGFs can enhance or inhibit the biological effects of IGFs. Osteoblasts express all six IGFBPs, and production varies with the cell system studied. Glucocorticoids decrease IGFBP-3, -4,and -5 in human osteoblasts. As IGFBP-5 is the only IGFBP known to enhance bone growth, this may be another possible mechanism by which glucocorticoids inhibit bone formation. The IGF and IGFBP effects are very complex, and further elucidation of the mechanisms by which glucocorticoids alter their interactions is a n important area of future investigation.

2. Local Factors a. Transforming Growth Factor+ TGF-P is secreted by osteoblasts in a latent, biologically inactive form that is incorporated into bone ECM. Osteoblasts not only produce TGF-P but also possess high-affinity receptors for it (Robey et al., 19871, providing the opportunity for autocrine stimulation of osteoblast replication. Latent TGF-P can be activated i n vitro by a number of agents, including acid pH, or by proteases such as plasmin or cathepsin (Lyons et al., 1988; Miyazono et al., 1988). TGF-P1 and 4 2 are homologous disulfide-linked homodimers of 25 kDa that have powerful effects on bone. These growthregulatory factors are present in the bone matrix in concentrations of 0.1 mg/kg dry weight. TGF-P can regulate gene expression of other locally active growth factors and cytokines. The local function of TGF-P may be very important in contributing to the differentiated activity of osteoblasts. It stimulates collagen synthesis and regulates gene expression of mRNA for p r o d (1)-chain collagen, osteonectin, alkaline phosphatase, fibronectin (Noda and Rodan, 1987), osteopontin (Noda et al., 19881, and osteocalcin (Noda, 1989). TGF-P increases the abundance of matrix proteins by stimulating their synthesis and inhibiting degradation. It is a potent stimulator of collagen and fibronectin synthesis and secretion in fibroblasts and osteoblasts (Ignotz and Massague, 1986; Sato et al., 19871, acting by increasing the mRNA for collagen and fibronectin (Ignotz et al., 1987). TGF-P also inhibits degradation of matrix proteins by decreasing the synthesis of matrix-degrading enzymes, as well as increasing the synthesis of protease inhibitors (Sporn et al., 1987).

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TGF-P promotes the differentiation of cells of the osteoblast lineage toward the mature osteoblast and the formation of new bone. Bone is the most abundant source of TGF-P in the body (Hauschka et al., 1986).TGF-P has been identified in culture medium conditioned by fetal rat calvaria (Centrella and Canalis, 1985), and confirmation of its osteoblast origin comes from the demonstration that fetal bovine osteoblasts transcribe TGF-P mRNA, synthesize and secrete the peptide, bear high-affinity cell surface receptors for it, and are mitogenically stimulated by TGF-P, probably through an autocrine mechanism (Robey et al., 1987).Stimulation of the replication of normal cells of the osteoblast lineage (Canalis, 1994)has been shown, as has growth inhibition of osteoblast-like osteogenic sarcoma cells (Noda and Rodan, 1987; Pfeilschifter et al., 1987) and of a clonal murine osteoblast-like cell line (Noda and Rodan, 1986). When fetal rat calvarial cells are exposed to active TGF-P, they respond with increased proliferation, while continued exposure impairs bone cell differentiation and the formation of mineralized nodules (Ghosh-Choudhury et al., 1994). The direction of TGF-P effects on proliferation of cells in the osteoblast lineage may depend on the stage of differentiation of the target cell as well as interactions with other factors. In addition to cell replication, matrix formation, and bone-related protein synthesis, TGF-P has been shown to induce fetal rat osteoblasts to produce hematopoietic cytokines. TGF-P induces IL-6 secretion in fetal rat bone cell cultures and enhances production of GM-CSF stimulated by PTH, IL-1, and bacterial endotoxin lipopolysaccharidein such cultures (Centrella et al., 1994). Additionally, TGF-P has major effects on human osteoblast cell protooncogene (c-fos and cjun) expression (Subramaniam et al., 1995). TGF-P also increases the capacity of osteoblasts to migrate unidirectionally (Pfeilschifter et al., 1989b). These data suggest that it may have an important chemotactic function in normal bone remodeling, attracting osteoblast precursors to sites of active bone resorption. TGF-P, injected subcutaneously adjacent to bone surfaces, causes a profound increase in new bone formation (Noda and Camilliere, 1989; Marcelli et al., 1990; Mackie and Trechsel, 1990). When TGF-P is administered by injection over the calvaria of mice daily for 3 days, bone width is increased 40% over the next month (Marcelli et al., 1990).This is initially woven bone, but it is later replaced by lamellar bone. Similar effects are seen when TGF-P is injected or infused directly into the marrow cavity of the femur. b. Znsulin-like Growth Factors I and 11. IGF-I and -11 are weak bone cell mitogens but have clear and potent stimulatory effects on the

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differentiated function of the osteoblast, as evidenced by an increase in osteocalcin and type I collagen synthesis in osteoblasts. As a result, IGFs increase bone matrix apposition rates and bone formation. IGFs also decrease collagen degradation and the expression of interstitial collagenase, suggesting a role in the preservation of bone matrix. IGFs enhance bone formation in uivo (Spencer et al., 1993). Mice with null mutation of type I IGF receptor have delayed skeletal development and delayed ossification (Liu et al., 1993). The anabolic properties if IGF-I and -11, their inhibitory actions on matrix degradation, and their abundance in bone tissue suggest that these factors play a central role in the maintenance of bone mass (Canalis, 1994). Several systemic factors can regulate IGF production. The anabolic effect on bone of intermittent administration of PTH has been attributed to expression of IGF-I. In organ cultures, transient exposure to PTH enhanced the production of IGF-I by three- to fourfold. Skeletal IGF production is only modestly dependent on growth hormone (Canalis, 1994), as growth hormone only marginally stimulates IGF-I production in osteoblasts (Delany et al., 1994b). 17P-Estradiol can increase IGF-I transcription in cells of the osteoblast lineage, while calcitriol has variable effects that differ with the model system studied. Glucocorticoids decrease IGF-I transcripts and protein concentrations in primary osteoblast cultures. Prostaglandin E, (PGE,) has been shown to increase IGF-I synthesis in osteoblast cultures, and promoter analysis of the IGF-I promoter revealed PGE,-responsive elements (Pash et al., 1995). In contrast, TGF-P, PDGF, and FGF inhibit IGF-I and -11 expression. Neither TGF-P, PDGF, or FGF stimulate differentiation of osteoblasts, while BMPs are potent stimulators of osteoblast differentiation. It is interesting to note that BMPs increase IGF-I production, suggesting that the ability of BMPs to stimulate IGF-I production (Canalis and Gabbitas, 1994) is linked to their ability to induce differentiation in osteoblasts. Regulation of IGF-I in bone is further complicated by the production of IGFBPs by osteoblasts. Osteoblasts express transcripts for the six known IGFBPs. Binding of IGF to one of these binding proteins can inhibit or potentiate the biological effect of IGF. Binding to IGFBP-1, for example, decreases the biological activity of IGF-I. Conversely, IGFBP-5 has been shown to increase bone formation and, thus, appears to enhance the effect of IGF-I. These observations are further complicated by the observation that growth factors such as TGF-P, PDGF, FGF, and BMP-2 inhibit synthesis of IGFBP-5 in bone cell cultures (Canalis and Gabbitas, 1995; Gabbitas and Canalis, 1999, while IGF-I and retinoic acid increase it (Dong and Canalis, 1995).

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Thus it appears that the effect of IGFs on bone is anabolic and that local regulation of IGFs in bone is highly complex. c. Bone Morphogenetic Proteins. BMPs are bone-derived peptides and members of the extended TGF-p superfamily. At least eight members are currently recognized. BMP-2 through BMP-8 share some TGF-p-related gene sequences. BMPs are synthesized by bone cells locally and stimulate the formation of ectopic bone when injected intramuscularly or subcutaneously into rodents (Urist, 1965). BMPs stimulate the replication and differentiation of normal cells of the osteoblast lineage and, in contrast to TGF-p, enhance the expression of the differentiated osteoblastic phenotype (Harris et al., 1995; Canalis, 1994). BMP-1, -2, -3, -4, and -6 are temporally expressed in primary cultures of fetal rat calvarial cells (Ghosh-Choudhury et al., 1994). BMP-2, -4, and -7 have been shown to induce differentiation of primitive mesenchymal cells into bone when implanted into subcutaneous tissue (Harris et al., 1995).BMP-2 accelerates differentiation in primary cultures of fetal rat calvarial cells, as demonstrated by an increase in expression of alkaline phosphatase and osteocalcin (Harris et al., 1995).BMP-3 decreases osteoclastic bone resorption and is chemotactic for monocytes (Cunningham et al., 1992).BMP-7 (osteogenicprotein-1) suppresses cell proliferation and stimulates the expression of markers characteristic of the osteoblast phenotype in rat osteosarcoma cells, but stimulates growth and differentiation in rat calvarial cultures (Maliakal et al., 1994). The precise role of BMPs in the bone remodeling process has yet to be determined. d . Heparin-Binding Fibroblast Growth Factors. Both acidic and basic FGFs are present in mineralized bone matrix and stimulate the replication of cells in the skeletal system, but do not increase the differentiated function of the osteoblast. Therefore, they may play an important role in bone repair when bone cell mitogenesis may be necessary (Canalis, 1994).TGF-p has been demonstrated to increase basic FGF expression in a mouse osteoblast cell line, while PTH and IL-1 had no effect (Hurley et al., 1994). Acidic and basic FGF have powerful stimulatory effects on bone formation in uivo. When injected locally over the calvaria of mice, FGF causes a 50% increase in bone thickness. When administered to ovariectomized rats, FGF blocked the associated bone loss and also increased trabecular connectivity and bone microarchitecture (Dunstan et a1 ., 1995). e. Platelet-Derived Growth Factor. PDGF stimulates the replication of bone cells but does not increase the differentiated function of the osteoblast (Hock and Canalis, 1994; Canalis, 1994). PDGF is produced by cells with the osteoblast phenotype (Graves and Owen, 1983; Graves

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et al., 19841, and osteoblasts have PDGF receptors (Xie et al., 1994). The A-chain homodimer of PDGF (PDGF-AA) is a less potent mitogen for osteoblasts cultured from fetal rat bone than those isoforms containing the B-chain subunits (PDGF-BB). Osteoblasts only synthesize PDGF A subunits, while the systemic form contains only PDGF B chains (Canalis et al., 1992). While some investigators have found that IL-la and TNF-a synergistically enhance the mitogenic effect of PDGF-AA, but not PDGF-BB, on osteoblasts, partly through enhanced binding (Centrella et al., 19921, others have demonstrated that IL-1 reduces PDGF receptor expression (Yeh et al., 1993) and reduces PDGF-AA binding to osteoblasts (Gilardetti et al.,1991). Since PDGF is a major component of platelets and is secreted during the platelet release reaction, its production a t sites of fracture healing may be important to the regeneration of bone. f . Prostaglandins. Prostaglandins have multiple effects on cells in the osteoblast lineage. They inhibit calvarial collagen synthesis in short-term cultures (Dietrich et al., 1976a,b), and, with long culture periods, collagen synthesis increases (Chyun and Raisz, 1984). Dogs injected locally with prostaglandins have increased bone volume (Ueno et al., 1985). PGE, stimulates the replication of cells in the periosteum and promotes collagen synthesis (Chyun et al., 19841, and increased collagen synthesis in response to PGE, has also been shown in clonal osteoblastic cells (Hakeda et al., 1985). In uiuo data support a stimulatory effect of prostaglandins on bone formation. The production of prostaglandins, predominantly PGE,, by osteoblasts has been well documented (Noda et al., 1982). PGE, increases IGF-I synthesis by osteoblasts as well (Pash et al., 1995). g . Interleukin-6. Murine stromal cells, human bone cells, and rat and mouse osteoblast-like cell lines activated with IL-1 and TNF-a all secrete IL-6 (Horowitz, 1993; Chaudhary et al., 1992), and IL-6 production has been demonstrated in normal human osteoblasts (Birch et al., 1993; Chaudhary et al., 1992). The secretion of IL-6 induced by these cytokines can be inhibited by treating the cells in uitro with estrogen and, to a lesser extent, with testosterone and progesterone (Girasole et al., 1992). Other investigators, however, have seen variable effects of sex steroids on IL-6 production by osteoblasts and stromal cells in similar experiments (Chaudhary et al., 1992; Rickard et al., 1992). Thus, it is possible that cytokine secretion by bone cells is downregulated by the addition of estrogen directly to osteoblasts and stromal cells, a finding that is consistent with the expression of estrogen receptors by osteoblasts (Komm et al., 1988; Eriksen et al., 1988). IL-6 production is also increased in bone cells by PTH (Feyen et al.,

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19891, IL-1, or TNF (Littlewood et al., 1991),suggesting that it may be a major mediator of osteoblast function. Specifically, PTH has been shown to induce IL-6 mRNA expression and protein secretion from a mouse osteoblast cell line as well as from primary rat osteoblast cultures (Greenfield et al., 1993). IL-6 can stimulate human osteoclastic bone resorption, and possibly the stimulatory effects on PTH, 1,25-(OH),D,, and IL-1 on bone resorption may in part be mediated through IL-6 production by osteoblasts. h. Tumor Necrosis Factor-a. TNF-a is a cytokine produced by macrophages and monocytes that has been implicated in the pathogenesis of osteoporosis, cancer-related cachexia, and septic shock. TNF-a stimulates bone resorption in uitro and causes hypercalcemia when administered to mice, The effects of TNF-a on bone formation are not as well understood and, from available literature, appear to be inhibitory. In uitro, TNF-a treatment of human osteoblasts inhibits expression of alkaline phosphatase and decreases collagen incorporation into developing matrix as well as mineralization of matrix without an effect on DNA synthesis (Panagakos et al., 1994). TNF-a treatment of various osteoblast-like cell lines inhibits PTH-stimulated rise in CAMP and free cytosolic calcium (Hanevold et al., 1993; Katz et al., 1992) by downregulating PTH receptors (Schneider et al., 1991).TNF-a inhibits calcitriol-stimulated synthesis of osteocalcin and inhibits vitamin D receptor number in a clonal rat osteosarcoma cell line (Mayur et al., 1993). In uiuo, infusion of TNF-a inhibits new bone formation stimulated by PTH-related protein (PTHrP) (Barengolts et al., 1994).Taken together, these observations indicate that TNF-a has an inhibitory effect on bone formation and may be one of the factors responsible for the uncoupling of bone resorption and bone formation observed in malignancy. i. Interleulzin-1 . IL-1 has growth-stimulating effects on osteoblast cells but inhibits differentiated function (Gowen and Mundy, 1986). IL-1 along with TNF impaired the responsiveness to PTH and PTHrP in cultures of clonal rat osteosarcoma cells (UMR-106)via downregulation of PTH receptors (Katz et al., 1992).Additionally, IL-1 and TNF-a had variable effects on osteoblastic expression of osteocalcin, alkaline phosphatase, and mineralized matrix depending on the system studied (Taichman and Hauschka, 1992). j . Interleukin-1 Receptor Antagonist. The IL-1 receptor antagonist (IL-lra)is a naturally occurring antagonist to IL-la and -1p that mediates its effects via binding to the IL-1 receptor (Hannum et al., 1990; Eisenberg et al., 1990). IL-lra decreases bone loss in ovariectomized rats (Kimble et al., 1994), and clinical studies suggest that secretion of

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IL-lra in the postmenopausal state may limit bone loss by decreasing IL-1 bioactivity (Pacifici et al., 1993). k. Interleukin-4. IL-4, a cytokine produced mainly by T lymphocytes, modulates the activity of lymphoid, hematopoietic, and mesenchymal cells. In uitro, IL-4 inhibits proliferation of osteoblast-like cells as well as enhances the expression of alkaline phosphatase stimulated by calcitriol (Riancho et al., 1993b). IL-4 alone inhibits expression of alkaline phosphatase and slows matrix mineralization in MC3T3 cells and primary cultures of rodent osteoblasts (Riancho et al., 1995). IL-4 stimulates monocyte colony-stimulating factor (M-CSF) expression, but not IL-1, IL-6, GM-CSF, or PGE,, by MC3T3 cells in these studies. In contrast, IL-4 increased hydroxyproline and osteocalcin accumulation and caused mineralization in cultured human osteoblastlike cells (Ishibashi et al., 1995).Additionally, IL-4 mediated osteoblast migration in a modified Boyden chamber system (Lind et al., 1995). In uiuo, transgenic mice that inappropriately express IL-4 under the direction of the lymphocyte-specific proximal promoter for the lck gene display severe osteoporosis of both cortical and trabecular bone (Lewis et al., 1993). The observed osteoporosis is characterized by decreased bone formation. Despite the observed disparate effects of IL-4 on osteoblast function in various in uitro systems, the in uiuo results in transgenic mice suggest that the major effect on bone is to inhibit osteoblast function. 1. Interleukin-11. IL-11 is a pleomorphic cytokine with biological activities that overlap with those of IL-6. Human osteosarcoma (SaOS-2) cells and primary human osteoblasts produce IL-11. Its production is enhanced in SaOS-2 cells after stimulation with IL-1, TGF-P, PTH, and PTHrP but not with IL-4, interferon-? or endotoxin (Elias et al.,1995). IL-11, along with IL-6, may be an important component of the cytokine network mediating osteoblast-osteoclast communication in bone remodeling (Manolagas and Jilka, 1995). m. Parathyroid Hormone-Related Protein. Tumor-produced PTHrP has a n established role as a mediator of malignancy-associated hypercalcemia. Its role in normal physiology is still under intense investigation, and studies demonstrate that PTHrP is important in normal bone development. Mice homozygous for the null mutation for the PTHrP gene are born with premature mineralization of normally cartilaginous areas (Karaplis et al., 1994). Furthermore, transgenic mice with targeted overexpression of PTHrP to proliferating and prehypertrophic chondrocytes by the mouse collagen type I1 promoter are born with marked foreshortening of the limbs and tail. Histological analysis of affected bones revealed a marked delay in chondrocyte

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maturation and endochondral ossification (Weir et al., 1995). In other experiments, PTHrP was detected in conditioned media from cultures of normal human bone cells, and PTHrP mRNA was detected in cells of the osteoblast lineage (Walsh et al., 1995). PTHrP production was inhibited by a variety of glucocorticoids in this system (Walsh et al., 1995). Taken together, these findings indicate that PTHrP is produced by normal bone cells and likely has an important role in the development of the normal skeleton. Determining this role is an important area of future investigation.

111. OSTEOCLASTS A. GENERAL CHARACTERISTICS OF THE OSTEOCLAST Osteoclasts are large, multinucleated cells that are the primary bone-resorbing cells. They are hematopoietic in origin and are formed by fusion of mononuclear precursors in the marrow. The osteoclast contains large quantities of tartrate-resistant acid phosphatase, a marker enzyme for the osteoclast, as well as hydrolytic enzymes, which are involved in the bone-resorptive process. Osteoclast formation and activity are regulated by factors produced from cells in the bone marrow microenvironment, including osteoblasts, stromal cells, and monocyte-macrophages. Characteristic features of the osteoclast include the presence of a ruffled border adjacent to areas in which bone is resorbed, as well as pleomorphic mitochondria and a prominent Golgi apparatus. In addition, osteoclasts have several unique features that distinguish them from macrophage polykaryons. These include the presence of calcitonin receptors, their ability to contract in response to calcitonin, cross-reactivity with several osteoclast-specific antibodies such as 121F (described by Oursler et al., 19851,and absence of Fc receptors. As noted earlier, osteoclasts are formed from hematopoietic precursors. This is based on transplantation studies in osteopetrotic rodents as well as in humans (Walker, 1972, 1973; Coccia et al., 1980; Sore11 et al., 19811, and in studies using bone marrow culture techniques (MacDonald et al., 1987; Takahashi et al., 1988;Kurihara et al., 1990b).The leading candidate for the earliest identifiable osteoclast precursor is the colony-forming unit-granulocyte-macrophage (CFU-GM), the granulocyte-macrophage progenitor. This cell, under the influence of a variety of cytokines, appears to differentiate to a more committed unipotent osteoclast precursor, l h i c h expresses calcitonin receptors

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(Takahashi et al., 1995a). These cells then fuse to form the multinucleated osteoclasts. The committed osteoclast precursor is postmitotic, expresses tartrate-resistant acid phosphatase, and cross-reacts with monoclonal antibodies that identify osteoclasts. Osteoclasts resorb bone through the production of proteolytic enzymes and secretion of hydrogen ions into the localized microenvironment under the ruffled border. This extracellular lysosome that is formed beneath the ruffled border results in degradation of collagen and calcified matrix. Hydrogen ion production in the osteoclast appears to be generated by the enzyme carbonic anhydrase 11, and these hydrogen ions are then pumped across the ruffled border by a vacuolar-type proton pump (Blair et al., 1989). Lysosomal enzymes are also released by the osteoclast and are highly activated in this acidic microenvironment. The bone resorption process results in formation of a resorption lacuna underneath the osteoclast. As the osteoclast then moves across the bone surface, additional resorption lacunae can be formed by a single osteoclast. Any process that interferes with the molecular mechanisms responsible for bone resorption or osteoclast formation results in osteopetrosis. A variety of techniques have been used to study osteoclast formation and function, including isolation of authentic osteoclasts from long bones, use of marrow culture techniques in which cells with a n osteoclast phenotype form, and use of giant cells isolated from giant cell tumors of bone as models for highly activated human osteoclast-like cells (Mundy and Roodman, 1987). Methods such as these have been employed to characterize osteoclasts and their precursors, as well as to determine the effects of local factors and systemic hormones on osteoclast formation and osteoclast activity. In addition, bone organ culture systems have been very useful for identifying factors controlling osteoclastic bone resorption, and several laboratories, including those of Lowik et al. (1989) and Burger and her associates (19821, have used fetal bone rudiments to study osteoclast biology and recruitment. Development of transgenic mice and mice in which gene function has been ablated by the techniques of homologous recombination has further helped to identify the role of specific factors in osteoclast activity and osteoclast formation. Studies in animals with osteopetrosis have also demonstrated some of the molecular defects responsible for impaired osteoclast function. These model systems have shown the importance of both locally acting factors and systemic hormones on osteoclast function and helped to elucidate the role that these factors play in both normal and pathological states, such as osteoporosis, Paget’s disease of bone, and multiple myeloma.

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B. SYSTEMIC HORMONES THATAFFECTOSTEOCLAST FUNCTION AND FORMATION 1. Parathyroid Hormone As noted earlier, PTH can either induce bone formation or inhibit osteoblast activity. When it is administered continuously, bone formation is suppressed and osteoclastic bone resorption is increased. Osteoclast precursors and osteoclasts have been shown to express PTH receptors (Teti et al., 1991; Hakeda et al., 1989),but the major mechanism that has been proposed for the action of PTH on osteoclasts has been an indirect one, in which PTH acts on the osteoblast, which in turn stimulates osteoclastic bone resorption. In patients with hyperparathyroidism, there is a loss of bone due to increased osteoclastic bone resorption accompanied by marrow fibrosis. PTH causes a marked increase in bone resorption in bone organ culture systems and stimulates osteoclast formation in both murine and human marrow culture systems (Takahashi et al., 1988; MacDonald et al., 1987).In addition, Lorenzo et al. (1986) have shown that PTH acts predominantly on a postmitotic cell. We have described an in uiuo model of osteoclast formation and demonstrated that PTH and PTHrP appear to act on the more differentiated osteoclast precursor, rather than the proliferative early osteoclast precursor (Uy et al., 1995b). This more differentiated precursor then fuses to form osteoclasts. Cytokines such as IL-1, TGF-a, TNF-a, and IL-6 can enhance the effects of PTHrP on osteoclast formation and osteoclastic bone resorption. IL-6,for example, appears to stimulate proliferation of early osteoclast precursors, and PTHrP then induces the differentiation and fusion of these precursors to form multinucleated osteoclasts (De La Mata et al., 1995). Furthermore, PTH and PTHrP enhance calcium reabsorption by the kidney. This enhanced renal calcium reabsorption contributes to the hypercalcemia seen in patients with malignancies, who have increased osteoclastic bone resorption due to overproduction of PTHrP by their tumors. Thus, although it is unclear if PTH acts directly or indirectly on osteoclasts, it has major effects on osteoclast formation and osteoclastic bone resorption in both normal and pathological states.

2. Calcitriol Vitamin D3 and its metabolites are potent stimulators of osteoclastic bone resorption and osteoclast formation. The most active metabolite, 1,25-(OH),D3, acts as a fusigen for committed osteoclast precursors (Takahashi et al., 1987). 1,25-(OH),D3 does not act on mature osteoclasts directly, since the mature osteoclast appears to lack vitamin D receptors (Narbaitz et al., 19831, and thus may have indirect effects on

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osteoclastic bone resorption (Feyen et al., 1989). 1,25-(OH),D, can induce IL-1 and IL-6 production by osteoblasts, factors that stimulate osteoclastic bone resorption. Furthermore, 1,25-(OH),D, increases calcium absorption from the gut and can act in conjunction with PTH to stimulate renal tubular calcium reabsorption, as well as enhance osteoclastic bone resorption stimulated by PTH. 3. Prostaglandins Prostaglandins are potent stimulators of osteoclastic bone resorption in bone organ culture systems and stimulate osteoclast formation in murine marrow cultures (Takahashi et al., 1988). However, PGE, inhibits osteoclastic bone resorption and formation in human systems (Chenu et al., 1990; Chambers et al., 1985).Chambers et al. (1985) have reported that PGE, induces osteoclastic contraction analogous to calcitonin and inhibits bone resorption by isolated osteoclasts. The effects of prostaglandins on osteoclast formation and osteoclastic bone resorption may be dose dependent and dependent on the assay system used. Tashjian and associates (1985) have found that a variety of factors that stimulate osteoclastic bone resorption in the mouse calvarial organ culture system do so by generating prostaglandins. Recently, Gallwitz et al. (1993)have shown that other arachidonic acid metabolites, such as the peptidoleukotrienes E, and D,, as well as 5-hydroxyeicosatetraenoic acid, stimulate isolated osteoclasts to resorb bone. These arachidonic acid metabolites may play an important role in bone resorption in areas of chronic inflammation. 4. Calcitonin

Calcitonin is a peptide hormone secreted by the parafollicular cells of the thyroid gland and is a potent inhibitor of osteoclastic bone resorption. It acts at multiple stages in the osteoclast lineage, including inhibition of osteoclast formation as well as the bone-resorbing capacity of mature osteoclasts. Calcitonin receptors are expressed on committed osteoclast precursors and appear to be a differentiation marker for the mature osteoclast (Takahashi et al., 1995a). Calcitonin downregulates expression of calcitonin receptors and osteoclast precursors in mature osteoclasts by inhibiting expression of the messenger RNA for its receptor (Takahashi et al., 1995a). Calcitonin acts on osteoclasts by stimulating adenylcyclase activity and CAMPaccumulation, which results in immobilization of the osteoclasts and their contraction away from the bone surface. Osteoclasts continually exposed to calcitonin escape from its effects. The mechanism responsible for this is unclear, but it may be due to the effects of calcitonin on expression of the calcitonin receptor. Calcitonin has been used as a therapeutic agent in

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patients with Paget’s disease and the hypercalcemia of malignancy, as well a s those with osteoporosis. In contrast to patients with hypercalcemia of malignancy, patients with Paget’s disease appear to be capable of responding to calcitonin for prolonged periods of time. However, the molecular mechanisms responsible for the increased responsivitiy of pagetic osteoclasts to calcitonin have yet to be defined. C. AUTOCRINE-PARACRINE FACTORS WITH OSTEOCLAST-STIMULATORY ACTIVITY

1. Znterleukin-1 IL-1 is a cytokine produced by monocyte-macrophages and marrow stromal cells. It can stimulate bone resorption in uitro and in uiuo. Several authors have shown that IL-1 induces bone resorption and osteoclast-like cell formation in murine and human marrow cultures (Gowen et al., 1983; Pfeilschifter et at., 1989a). Uy et al. (1995a) have used a n in uiuo model of osteoclast formation to examine the effects of IL-1 on the different stages of osteoclast differentiation. They used IL-1 as a n osteotropic factor to delineate the cellular mechanisms responsible for enhanced osteoclast activity stimulated by IL-1. IL-1 induced hypercalcemia and enhanced the growth and differentiation of CFU-GM, the earliest identifiable osteoclast precursor; increased the number of more committed mononuclear osteoclast precursors; and stimulated mature osteoclasts to resorb bone. The effects of marrow stromal cells on osteoclast formation are also in part mediated by IL-1. Takahashi et al. (1995b) established a human bone marrow stromal cell line (Saka cells) by infecting marrow-adherent cells from semisolid marrow cultures with a recombinant adeno/simian virus-40 virus. Coculture of Saka cells with human marrow mononuclear cells enhanced formation of osteoclast-like multinucleated cells in human marrow cultures. These osteoclasts expressed calcitonin receptors and formed resorption lacunae on dentine. Polymerase chain reaction analysis of the Saka cells detected expression of mRNAs for IL-lp. Addition of neutralizing antibodies to IL-1p blocked the effects of Saka cells on osteoclast-like cell formation. IL-1 has been implicated in the increased bone loss seen in several pathological states. It is produced by several tumors associated with hypercalcemia, such as squamous cell carcinoma and lymphoma (Fried et al., 1989; Sat0 et al., 1989). Freshly isolated marrow cells derived from some patients with myeloma produced IL-lp, and the bone-resorbing activity present in culture media from these marrow cell iso-

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lates could be neutralized by IL-lp antibodies (Kawano et al., 1989; Cozzolino et al., 1989). Kitazawa et al. (1994) have demonstrated that IL-1 plays a direct role in mediating the effects of ovariectomy on osteoclastogenesis and bone resorption. Ovariectomy increased the bone marrow cell secretion of IL-1 and the formation of TRAP(+) osteoclast-like cells in bone marrow cultures treated with vitamin D, and in uiuo. The effects of ovariectomy on osteoclast formation in uiuo and in uitro were blocked by treatment with a n anti-IL-lra. The mechanisms mediating the effects of IL-1 on osteoclasts are not completely understood. In an in uiuo model system designed by Boyce et al. (1989a,b), administration of IL-la and IL-1p systemically stimulated bone resorption in mice. Treatment of the mice with indomethacin partially inhibited the effects of IL-1, suggesting that part of the effects of IL-1 were mediated by prostaglandin. Thus, IL-1 is a potent osteotropic factor that stimulates osteoclasts at all stages of differentiation and induces bone resorption both in uiuo and in uitro. Its role in disease states, as well as the biology of its effects on the osteoclasts, remain to be completely elucidated. 2. Colony-Stimulating Factors

Colony-stimulating factors are hematopoietic growth factors that induce clonal growth of hematopoietic progenitors in uitro and in uiuo. They are produced by macrophages, stromal cells, endothelial cells, and T lymphocytes in the marrow microenvironment. Since the osteoclast is hematopoietic in origin, it is not surprising that these factors may act as stimulatory factors for the osteoclast as well. Studies in animals and patients with osteopetrosis have shown that bone marrow transplantation can cure osteopetrosis (Walker, 1975a,b; Coccia et al., 1980; Sore11 et al., 1981). The oplop mouse, in which the M-CSF gene is mutated, has greatly reduced numbers of macrophages and osteoclasts. I n uiuo, exogenous M-CSF has been shown to be necessary for the generation of osteoclast-like cells in cocultures of hematopoietic and stromal cells from op/op osteopetrotic mice. I n uivo administration of M-CSF restores osteoclastogenesis and bone resorption in oplop mice (Morohashi et al., 1994). The effects of other colony-stimulating factors, including GM-CSF, are not clear as those for M-CSF. GM-CSF stimulates the growth of osteoclast precursors and induces osteoclast formation when human bone marrow cultures are treated sequentially with GM-CSF followed by 1,25(OH),D, (MacDonald et al., 1986). However, GM-CSF by itself

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has no effect on osteoclastic bone resorption, and it has been reported to inhibit osteoclastic bone resorption in murine marrow cultures (Shuto et at., 1994). Takahashi and associates (1991) have examined the effects of colony-stimulating factors on murine osteoclast formation in uitro in a coculture system, and have shown that all these factors can stimulate the growth of osteoclast precursors but, when added simultaneously with 1,25(OH),D3, inhibit its effects on osteoclast formation. 3. Transforming Growth Factor-a

TGF-a is a polypeptide of 5700 Da that is partially homologous t o epidermal growth factor (EGF). It is produced by several solid tumors associated with the hypercalcemia of malignancy and can stimulate osteoclastic bone resorption in murine organ cultures (Ibbotson et al., 1985). The effects of TGF-a are mediated through the EGF receptor and are independent of prostaglandin synthesis (Yates et al., 1992). TGF-a induces osteoclastic bone resorption and hypercalcemia in nude mice and increases osteoclast-like cell formation in human marrow cultures (Yates et al., 1992; Takahashi et al., 1986a). Hiraga et al. (1995) demonstrated the presence of TGF-a by immunohistochemistry in areas of bone adjacent to osteoclast activation and bone resorption induced by a metastatic human melanoma cell line in nude mice. Therefore, TGF-a can stimulate osteoclast formation and bone resorption both in uitro and in uiuo. 4. Tumor Necrosis Factor

Both TNF-a and TNF-P (lymphotoxin) markedly stimulate the formation of osteoclast-like multinucleated cells in human marrow cultures (Pfeilschifter et al., 1989a). TNF can also affect the activity of mature osteoclasts. Thomson et al. (1986, 1987) have shown stimulation of bone resorption by mature osteoclasts when these cells were incubated with IL-1 or TNF and cocultured with osteoblastic cells. TNF potentiates the effects of IL-1 on osteoclast formation (Pfeilschifter et al., 1989a); therefore, its effects on osteoclasts may be in part mediated by other cytokines. Garrett et al. (1987)demonstrated that myeloma cell lines produced lymphotoxin in uitro, and neutralizing antibodies t o lymphotoxin blocked the bone resorption in bone organ cultures induced by media conditioned by these cells. However, increased levels of TNF-P have not been found in an in uiuo model of human myeloma bone disease (Alsina et al., 1995). TNFs appear to

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stimulate both proliferation and differentiation of precursors for osteoclast-like cells to osteoclasts and may be involved in the pathogenesis of hypercalcemia of malignancy.

5 . Interleukin-6 IL-6 is a 26,000-Da cytokine produced by marrow stromal cells, monocyte-macrophages, osteoclasts, and osteoblasts. It induces osteoclast formation and bone-resorbing activity of preformed osteoclasts (Lowik et al., 1989; Kurihara et al., 1990a). However, it does not appear, by itself, to be a potent osteotropic factor in murine systems in uiuo. IL-6 potentiates the effect of other hormones such as PTHrP on calcium homeostasis and osteoclastic bone resorption in uiuo, as demonstrated by De La Mata et al. (1995). IL-6 production by bone and marrow stromal cells is suppressed by 17P-estradiol in uitro. In mice, estrogen loss with ovariectomy increased the number of CFU-GMs, enhanced osteoclast development in ex uiuo cultures of marrow, and increased the number of osteoclasts in trabecular bone. These changes were prevented by administration of a n antibody to IL-6 (Jilka et al., 1992). These findings suggest that IL-6 may be involved in the increased bone resorption in postmenopausal osteoporosis, with estrogen loss resulting in a n IL-6-mediated stimulation of osteoclasts. However, others have implicated IL-1 and/or TNF-a as potential mediators for the bone loss seen with ovariectomy (Kimble et al., 1994). IL-6 may also act as a n autocrine-paracrine factor in Paget’s disease (Roodman et al., 1992). Osteoclast-like multinucleated cells formed in human marrow cultures from patients with Paget’s disease actively release IL-6 into their conditioned medium, and this medium stimulated osteoclast-like cell formation in normal marrow cultures. Furthermore, patients with Paget’s disease, but not normal subjects, have elevated levels of IL-6 in their marrow plasma and their peripheral blood. We examined the effects of antisense constructs to IL-6 on the boneresorbing capacity of purified giant cells from giant cell tumors of bone to help define the role of IL-6 in human osteoclastic bone resorption (Reddy et al., 1994). IL-6 levels were elevated in conditioned medium from highly purified giant cells. Treatment of these giant cells with IL-6 antisense constructs or neutralizing antibodies to IL-6 caused a fourfold decrease in IL-6 levels, and significantly decreased the number of resorptive lacunae formed and the area of the dentine resorbed. These observations demonstrate that IL-6 may play a n important role in the bone-resorptive process of human osteoclasts.

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Interestingly, IL-6 has been found to induce a marked loss of calcitonin-binding sites on normal T lymphocytes at concentrations known to be active on bone metabolism (Body et al., 1994). IL-6 may also play an important role in other states of increased bone destruction, such as multiple myeloma bone disease and Gorham-Stout disease, or disappearing bone syndrome, where it has been implicated by Devlin et al. (1995a). 6. Annexin IZ Identification of the production of autocrine factors by osteoclasts represents an important addition to our understanding of normal osteoclast formation and activity. Takahashi et al. (1994)have prepared a mammalian cDNA expression library generated from highly purified human osteoclast-like multinucleated cells formed in long-term bone marrow cultures and screened this library for autocrine factors that enhance osteoclast-like cell formation. In the initial screening annexin I1 was identified, and purified recombinant annexin I1 significantly increased osteoclast-like cell formation in human bone marrow cultures in the absence of 1,25(OH),D,. It also enhanced the bone-resorptive capacity of 1,25(OH),D3 in bone organ cultures. Interestingly, annexin I1 mRNA was expressed at high levels in RNA isolated from highly purified giant cells from osteoclastomas, human osteoclast-like cells, and pagetic bone. Nesbitt and Horton (1995) have reported that annexin I1 is also expressed on the surface of osteoclasts, and inhibition of annexin I1 with an antibody to it blocked bone resorption by isolated osteoclasts. Devlin and associates (1995b)have demonstrated that annexin I1 stimulates the proliferation of osteoclast precursors in human marrow cultures and enhances the effects of GM-CSF on the growth of these precursors. Annexin I1 appears to be a proliferative factor that enhances the growth of osteoclast precursors, as well as plays a role in osteoclastic bone resorption. D. LOCAL INHIBITORY FACTORS 1. Transforming Growth Factor+

TGF-P is one of the key factors involved in coupling bone formation to previous bone resorption (Mundy, 1991).It is secreted by osteoblasts and osteoclasts and may act as an autocrine factor stimulating osteoblastic bone formation through enhanced chemotaxis, proliferation, and differentiation of committed osteoblasts.

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TGF-P is secreted as a dimer composed of 12.5-kDa subunits noncovalently associated with one or more polypeptides to form a higher molecular weight latent complex. Latent TGF-P can be experimentally activated by proteinase treatment or denaturation to remove the binding proteins. Oursler (1994) has shown that osteoclasts expressed mRNA for TGF-P and that latent TGF-P that is secreted may be activated by osteoclasts. She concluded that osteoclasts may secrete proteinases that activate latent TGF-P into the extracellular space, and that TGF-P may be a n autocrine factor for osteoclasts as well. Similarly, Pfeilschifter and Mundy (1987) have reported that osteoclasts can activate latent TGF-P. TGF-(3 has been shown to be a potent inhibitor of osteoclastic bone resorption by modulating both osteoclast migration and osteoclast differentiation (Chenu et al., 1988). It probably also plays a role in the regulation of the proliferation of osteoclast progenitors in uiuo, since it was shown by Dieudonne et al. (1991) to prevent the increase in the number of TRAP( + 1 multinucleated osteoclast-like cells when administered systemically to ovariectomized rats. Similarly, Chenu et al. (1988) have shown that TGF-P inhibits both the proliferation and fusion of human osteoclast precursors. 2. y-Interferon y-Interferon is a potent inhibitor of bone resorption in uitro (Gowen and Mundy, 1986), and suppresses the formation and maturation of osteoclasts (Takahashi et al., 198613).Tohkin et al. (1994) examined the effects of y-interferon on humoral hypercalcemia in nude mice bearing lower jaw tumors, in which PTHrP is responsible for inducing hypercalcemia. Mice were injected with y-interferon for 5 days before the establishment of hypercalcemia, and the increase in plasma calcium concentration was delayed. y-Interferon also abolished the formation of multinucleated osteoclast-like cells from bone marrow cells of these mice in uitro. The data suggest that y-interferon suppresses the formation of osteoclasts, resulting in prolonged decrease in plasma calcium concentration. Gowen and Mundy (1986) have also shown that y-interferon blocks the bone-resorbing effects of IL-1 and TNF. y-Interferon appears to be a more effective inhibitor of bone resorption stimulated by IL-1 and TNF than bone resorption stimulated by PTH or 1,25(OH),D3. Similarly, Kurihara and Roodman (1990) have shown that other interferons can also inhibit osteoclast formation in human marrow cultures, suggesting that the interferons as a class are inhibitors of bone resorption.

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3. Interleukin-4 IL-4 is a product of activated T cells with effects on both immunological and hematopoietic processes. Shioi et al. (1991)reported that IL-4 inhibited the formation of osteoclasts from murine bone marrow cells cocultured with stromal cells, and Watanabe et al. (1990) showed that IL-4 inhibited bone resorption in organ cultures. Others have reported similar results (Riancho et al., 1993a). Nakano et aZ. (1994) examined the in uiuo effects of IL-4 on spontaneous and stimulated mouse osteoclast formation. EC-GI cells, which produce PTHrP and IL-1, were explanted into nude mice. After the mice became hypercalcemic, treatment with a continuous infusion of IL-4 returned the calcium levels to normal. Histomorphometric analysis revealed that IL-4 inhibited osteoclast formation in these mice, with a decrease in osteoclastic surface and in the number of osteoclasts per normal bone surface. Furthermore, transgenic mice overexpressing IL-4 develop an osteopenic syndrome that may be likened to osteoporosis (Lewis et al., 1993). Thus, IL-4 exerts inhibitory effects on osteoclasts and osteoblasts, both in viuo and in uitro. IV. SUMMARY Systemic hormones and cytokines play important roles in regulating both osteoblast and osteoclast activity. These cytokines can have either positive or negative effects on the growth and differentiation of bone cells. These effects appear to be dependent on the model systems use to assess them, as well as the species tested. In the near future, other autocrine-paracrine factors will be identified that enhance osteoblast and osteoclast activity, and model systems should be available to further delineate their effects on cells in the osteoblast lineage. Use of transgenic mice with genes targeted to the osteoblast and osteoclast may further reveal the mechanisms responsible for the growth and differentiation of these cells, as well as produce immortalized cell lines that more accurately reflect the cell biology of the osteoclast and osteoblast in viuo. REFERENCES Alsina, M., Boyce, B., Devlin, R., Anderson, J. L., Craig, F., Mundy, G.R., and Roodman, G.D. (1995). Development of an in vivo model of human multiple myeloma bone disease. Blood, in press. Barengolts, E. I., Lathon, P. V., Lindh, F., and Kukreja, S. C. (1994).Cytokines may be responsible for the inhibited bone formation in hypercalcemia of malignancy. J. Bone Miner. Res. S(Supp1 l),S138.

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Bellows, C. G., Aubin, J. E., Heersche, H. M. N., and Antosz, M. E. (1986). Mineralized bone modules formed in vitro from enzymatically released cat calvaria cell populations. Calcif. Tissue Int. 38, 143-154. Birch, M. A., Ginty, A. F., Walsh, C. A., Fraser, W. D., Gallagher, J. A., and Bilbe, G. (1993). PCR detection of cytokines in normal human and pagetic osteoblast-like cells. J . Bone Miner. Res. 8, 115-1162. Blair, H. C., Teitelbaum, S. L., Ghiselli, R., and Gluck, S. (1989). Osteoclastic bone resorption by a polarized vacuolar proton pump. Science 245, 855-857. Body, J. J., Fernandez, G., Lacroix, M., Vandenbussche, P., and Content, J. (1994). Regulation of lymphocyte calcitonin receptors by interleukin-1 and interleukin-6. Calcif. Tissue Int. 55, 109-113. Boyce, B. F., Aufdemorte, T.B., Garrett, I. R., Yates, A. J. P., and Mundy, G. R. (1989a). Effects of interleukin-1 on bone turnover in normal mice. Endocrinology 123,11421150. Boyce, B. F., Yates, A. J. P., Mundy, G. R. (1989b). Bolus injections of recombinant human interleukin-1 cause transient hypocalcemia in normal mice. Endocrinology 125,2780-2783. Burger, E. H., Van der Meer, J. W. M., Gevel, J. S., Gribnau, J. C., Thesingh, C. W., and van Furth, R. (1982). In vitro formation of osteoclasts from long-term cultures of bone marrow mononuclear phagocytes. J . Exp. Med. 156, 1604-1614. Canalis, E. (1993). Systemic and local factors and the maintenance of bone quality. Calcif. Tissue Int. 53(Suppl), S90-S93. Canalis, E. (1994). Editorial: Skeletal growth factors and aging. J. Clin. Endocrinol. Metub. 78, 1009-1010. Canalis, E., and Gabbitas, B. (1994). Bone morphogenetic protein 2 increases insulinlike growth factor I and I1 transcripts and polypeptide levels in bone cell cultures. J. Bone Miner. Res. 9, 1999-2005. Canalis, E., and Gabbitas, B. (1995). Skeletal growth factors regulate the synthesis of insulin-like growth factor binding protein-5 in bone cell cultures. J.Biol. Chem. 270, 10771-10776. Canalis, E., and Lian, J. B. (1985). 1,25-Dihydroxyvitamin D2 effects on collagen and DNA synthesis in periosteum and periosteum-free calvaria. Bone 6, 457-460. Canalis, E., Varghese, S., McCarthy, T. L., and Centrella, M. (1992). Role of platelet derived growth factor in bone cell function. Growth Regul. 2, 151-155. Canalis, E., Pash, J., Gabbitas, B., Rydziel, S., and Varghese, S. (1993). Growth factors regulate the synthesis of insulin-like growth factor-I in bone cell cultures. Endocrinology 133, 33-38. Centrella, M., and Canalis, E. (1985). Transforming and non-transforming growth factors are present in medium conditioned by fetal rat calvariae. Proc. Nutl. Acad. Sci. U.S.A.82,7335-7339. Centrella, M., McCarthy, T. L., Kusmik, W. F., and Canalis, E. (1992). Isoform-specific regulation of platelet-derived growth factor activity and binding in osteoblast-enriched cultures from fetal rat bone. J . Clin. Invest. 89, 1076-1084. Centrella, M., Horowitz, M. C., Wozney, J. M., and McCarthy, T. L. (1994). Transforming growth factor-p gene family members and bone. Endocr. Rev. 15, 27-39. Chambers T. J.,McSheehy, P. M. J., Thomson, B. M., and Fuller, K. (1985).The effect of calcium-regulating hormones, prostaglandins on bone resorption by osteoclasts disaggregated from neonatal rabbit bones. Endocrinology 116, 234-239. Chaudhary, L. R., Spelsberg, T. C., and Riggs, B. L. (1992). Production of various cytokines by normal human osteoblast-like cells in response to interleukin-1 beta and

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